Molecular imprinting refers to a templating technique for producing inverse replicas of individual molecules in network polymer. This approach has been used to generate porous materials exhibiting receptor-like affinity for a large variety of template structures. Commonly referred to as plastic antibodies, these can now be produced of a similar size and featuring binding properties resembling antibodies. Their ability to function in complex environments including biological environments in vivo has therefore considerably expanded their scope of applications, mainly as antibody substitutes in assays, sensors, separations and as a new class of drugs or therapeutic tools. The development of new and improved methods for producing these receptors is hence an urgent goal, from both an industrial and societial perspective.
Most examples of high fidelity molecular imprinting have been demonstrated using highly cross-linked organic polymers as the imprinting matrix. Free radical polymerization of functional vinyl monomers with an excess of cross-linking divinyl monomers in presence of a porogen, usually a solvent for the monomers, has for long been the method of choice to produce these materials. To form the binding sites, a template or template-monomer adduct is added prior to polymerization. Subsequent removal of the template results in a porous organic polymer material equipped with binding sites for the template ion or molecule.
One recurring problem associated with molecularly imprinted polymers (MIPs) is viewed when testing the reuptake of template to the empty binding sites. Here, a strong dependence of the partition coefficient on the sample load is commonly seen. The origin behind this effect is in most cases a heterogeneous distribution of binding sites. In non-covalent imprinting, two effects contribute primarily to the binding site heterogeneity. Due to the amorphous nature of the polymer, the binding sites are not identical, somewhat similar to a polyclonal preparation of antibodies. The sites may, for instance, reside in domains with different cross-linking density and accessibility. Secondly, this effect is reinforced by the incompleteness of the monomer-template association.[5] In most cases the major part of the functional monomer exists in a free or dimerized form, not associated with the template. As a consequence, only a part of the template added to the monomer mixture gives rise to selective binding sites. The low yield of binding sites results in a strong dependence of selectivity and binding uptake on sample load.
Template occlusion is another recurring problem in traditional molecular imprinting. Typically a small fraction of the template added to the monomer mixture remains entrapped or bound in the polymer matrix which can result in bleeding—a process detrimental when using the MIPs as enrichment phases in trace analysis. Moreover, template recovery is not straightforward requiring multiple purification steps. This is unpractical and costly in cases where expensive templates are used.
Finally, even when approaches are found successfully addressing each of the aforementioned problems, the final imprinting technique should be economically attractive, and scalable i.e. allowing facile scale up to meet industrial demands. Since developing a new MIP is an interative process, several MIPs needs to be synthesized and screened before the right one is found. This optimization requires parallel synthesis protocols and miniaturization which are other criteria for a practical imprinting technique.
Several approaches have been assessed in order to overcome the aforementioned problems. Hardly avoidable, the kinetically controlled formation of the polymer network leads to a statistical distribution of binding site microenvironments. One way to overcome this problem is to use polymer matrices formed by thermodynamically controlled reversible polymerization reactions. Controlled radical polymerization (CRP) may offer benefits in this regard.[8] CRP distinguish itself relative conventional radical polymerization in the low active radical concentration and the life time of the growing radical. This allows the preparation of polymers with predefined molecular weights, low polydispersity, controlled composition and functionality. Other ways to reduce the polyclonality of imprinted sites have been suggested. A particularly simple approach in this context is to thermally cure or anneal the polymers which was anticipated to cause polymer chain relaxation leading to enhanced thermodynamic control of the binding site formation. Another approach was the use of high pressure polymerizations anticipating that this would stabilize the monomer template complexes and thus reduce heterogeneity caused by this factor.
Alternatively, the template may be confined by immobilization at a solid liquid interface or liquid liquid interface.[12] This technique is referred to as interfacial imprinting and presumably leads to a more defined microenvironment due to the restricted template mobility during polymerization and subsequent removal of the poreforming phase.
Finally, nanoparticles or nanogels produced by precipitation, emulsion or graft polymerization, and thin film materials may exhibit more homogenous binding sites due to the spatial restrictions imposed by the limited film thickness or microgel radius. Furthermore by reducing the size of the MIP to form particles in the nanometer range, for very small particles referable to as soluble MIPs, MIPs with on average one site per particle can be produced. In such systems heterogeneity is mainly due to the non-equivalence of sites between the particles (cf. polyclonal antibodies) and as polyclonal antibodies the particles can be fractionated and enriched by affinity chromatography. Miniemulsion polymerization protocols can be used for interfacial imprinting of such nanoparticles, hence combining the advantages of the nanoparticle format, and interfacial imprinting.
In spite of the above individually promising results, none of the reported methods address the remaining problems i.e. 1) template occlusion and recycling and 2) process scalability and 3) parallelism and miniaturization. There is therefore a need for new techniques allowing simultaneously interfacial imprinting, nanoparticle production, template recycling and parallelism and scalability.